1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modem molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Ulhinann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6, 12 (1995); and Antisense Research and Applications (Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72, 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34, 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22, 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modem approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28, 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23, 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramnidite chemistry. Jager et al., Biochemistry 27, 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Antl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by each of the foregoing methods involves the same generalized protocol. Briefly, this approach comprises anchoring the 3'-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Desired internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the 5'-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 .mu.mol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4, 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis (Padmapriya et al., supra; Ravikamar et al., Tetrahedron 50, 9255 (1994); Theisen et al., Nucleosides & Nucleotides 12, 43 (1994); and Iyer et al., Nucleosides & Nucleotides 14, 1349 (1995)) and isolation (Kuijpers et al. Nucl. Acids Res. 18, 5197 (1990); and Reddy et al., Tetrahedron Lett. 35, 4311 (1994)) of oligonucleotides.
There has been recent interest in stereodefined phosphorothioate antisense oligonucleotides. Recently, enzymatic synthesis of "all [R.sub.p ]" PS-oligos was achieved using nucleoside 5'-[S.sub.p ]-.alpha.-thiotriphosphates in conjunction with DNA polymerase. Tang et al., Nucleosides Nucleotides 14, 985 (1995). But enzymatic methodology is not as yet amenable to large-scale work, and in addition, does not provide [S.sub.p ] PS-oligos.
Attempts to achieve stereoselective synthesis of PS-oligonucleotides using phosphoramidite chemistry in conjunction with diastereomerically pure phosphoramidites has not been successful. Thus, when pure [R.sub.p ] or [S.sub.p ] nucleoside .beta.-cyanoethyl phosphoramidites were employed in the synthesis of PS-oligos, a mixture of [R.sub.p ] and [S.sub.p ] PS-oglios were produced. For a review, see: Beaucage and Iyer, Tetrahedron 48, 2223 (1992). Presumably, this was because of the 1H-tetrazole-mediated epimerization of the phosphorous center during the coupling step of oligonucleotide synthesis. Nucleoside oxathiaphospholane is a novel synthon developed by Stec and coworkers to prepare short, but stereodefined PS-oligos. Koziolkiewicz et al., J. Antisense Nucl. Acid Drug. Dev. 7, 43 (1997) and references therein. However, this approach requires: (a) prior separation on the individual P-diastereomers (b) need longer coupling reactions compared to standard phosphoramidite chemistry, and (c) the presence of unprotected phosphorothioate functionality generated during the coupling reaction may potentially cause the formation of side products during oligonucleotide synthesis. An interesting stereoselective route towards TpsT using indoloxazaphosphorine intermediate was disclosed recently. Wang and Just, Tetrahedron Lett 38, 3797 (1997) and references therein.
Iyer et al., J. Org. Chem. 60, 5388 (1995) reported the use of oxazaphospholidines as alternate synthons in oligonucleotide synthesis. Iyer et al., Tetrahedron Asymmetry 6, 1051 (1995) reported the stereoselective synthesis of dinucleoside phosphorothioates using a related oxazaphospholidine. The increased reactivity of the P (III) center towards nucleophilic attack and the conformational restraint imposed by the 5-membered ring in the oxazaphospholidine was expected to out-compete the tetrazole-catalyzed epimerization at the P-stereocenter during coupling. Additionally, the presence of the chiral auxiliary in the ring could provide the requisite facial bias in the nucleophilic attack by the support-bound nucleoside on the oxazaphospholidine.